The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K{+}-Cl- co-transporters

Precise homeostasis of intracellular Cl- is achieved via the coordinated activities of the Cl- influx and efflux. We demonstrate that the WNK activated SPAK/OSR1 kinases known to directly phosphorylate and stimulate the sodium-potassium co-transporters (N[K]CC), also promote inhibition of the potassium-chloride co-transporters (KCCs) by directly phosphorylating a recently-described C-terminal threonine conserved in all KCC isoforms (Site-2 [Thr1048]). First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Second, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Third, in embryonic stem cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of 86Rb+ uptake that was not markedly stimulated further by hypotonic low Cl- conditions, consistent with KCC3A activation. Fourth, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity, while a double alanine KCC3A Site1/2 mutant is constitutively active. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr5 KCC1/KCC3 and Thr6 KCC2/KCC4), and a previously recognised KCC3-specific...

Source: BJ Signal - January 7, 2014 Category: Biochemistry Authors: P de los Heros, D R. Alessi, R Gourlay, D G. Campbell, M Deak, T J. Macartney, K T. Kahle, J Zhang Tags: BJ Signal Source Type: research